E: I, E:CT R 0I,\-T IC
-1T IOS
I) I SS0 C I
BY
a.
SD T OSIC
E F FE CT
F. C L I K I . :
I. INTRODUCTORY T h e object the writer had in view in imdertaking the investigation, the results of n-liich are presented in this paper>\\.as to deteriiiiiie approximately the relati\-e and absolute toxic properties of a fen- deleterioiis agents as sho\vii 1 3 ~ . their influence on tlie spores, the inj.ceiiuiii, aiicl the fructification of certain of the mold fiingi. In recent years the stndy of pinnt patliology has come to l x one of the most important in the nhole range of botanical research. It seems desirable, therefore, that all possihle light should be thrown on the toxic properties of the various agcnts used in coiiiliatiiiy fiiiigoiis pests. It is also \-er!. clt.sirable f r o i i i 3 scientific poiiit of \-ien-, to throw all light possiible on the problein as to the rlriitcut or ~ R J I / ) ~ f ~ / ~ i i i c ito ~ tthe . s influence of \vliicli the toxic properties of the coiiipoiiiid are to lie attributed. It has heeii tlie aim of the writer to suppleinelit tlie excelleiit irork of Drs. Kalilenliei-g, Ti-lie,' and Healtl- 011 plinnerogams aiid Drs. Kronig aiid 1'3111: 011 llacteria bj. appl!.iiig tlie tl:eor!. of tlie electrol\.tic dissociation of the niolecule to the stud!. of the physiolog?. of the effect of toxic agent.; 011 the inold fiiiigi. 11. METHODS I. .Yc/pl.f/'oii /!/- F?ii.iii.s. - In selecting forins t h e follon.iiig points Lvcre gil-eii particular consideration : i a i , c y / / l u i . i f j , ,!/' ~ f , i , / i ~ i i ~ ((11) ~ f iabilitj. / i / ~ , to grow i i o r i i t l ~ / ( i ,in a liqnitl i~iediuiii, ( c )ability tcJ friiit iiorniall~.iii a saturated atiiiospliere. & \ h e r
'
I k l l . Gaz.
' I b i ~ l2. 2 .
22) h i
':r s y h : .
~ 1 8 9 '6. %?it. i. Hygiene 11. Iiifect. 125
25, I
rS9;"l.
csperiiiientatioii with a large number of forms the following n-ere chosen as being x-er?.well suited for the n-ork : ,Is$ci;qi/lus f l o x i s Link, .Sfri,i~~iizntoc;ilsf2j. u ~ ' g r nv. Tieghem, Edorcphnlziiiz olhiduui Saccardo, and Pc/zz'~-iilirini g / n z ~ c z ~ i /1,ink. / h'otqifis ;~/c[yor-l's Fr.was afterwards chosen as a fifth form, it being entirely satisfactor!. in regard to geriiiiiiation aiid mj.celial development atid especiall?. desirable on account of its semi-parasitic habit. I t , howe\-er, failed to fruit in cell cultures. Pure cultures of these fiiiigi were obtained and renewed from week to \veek. Tlie spores used iii inoculating the cultures in the esperiniental work were taken from freFh tubes in which the funxiis had heen growing sei-en to fourteen da?.s. -4 solid mediiuii made b!, adding twelve grams of agar to a liter of sugar beet iiifiisioii n-as found to he T-erj. satisfactor!. for stock cultures. 2 . S ~ / c c ~ i o i(f / -1icdizLm.- T h e object heiiig to test the effect of the deleterions agents on the fungi when growing in as iiearl!. normal conditions as possible, the selection of a suitable iiiediiiiii became a r.er!- important matter. Many prelitiiinarjcultures were made with varions media, including distilled \vater, infnsions of potato, celer!., sugar beet, prune, and bean (steiiis, pods, and mature seeds ) $ besides various others coniponncled from inorganic salts, sugars, asparagin, etc. T h e results in germination and development were l-er?. varied. IIi distilled water .Sfc'i-z,oiiinfI)r~isfI'saiid P~/zici//z'zim failed to germiiiate in 2 4 hours and none of the five foriiis germinated more than 40 percent. Mycelial de\.elopment was meager, and fruiting generally riil. T'ery minute quantities of the deleterious agents were found to inhibit germination, hut the &nz%+ol'izf of the spore was found. in tlie case of dicliloracetic acid, potassiuiii li?droxide, and cobaltous sulphate, to he tlie same in pure water 3s iii the niedium finall!. selected. Tlie media coiiipouiided froiii salts, sugars, etc., were more satisfactory ; the vegetable infusions, however, were superior to all others. ,111infusion of the sugar beet was idtimatel?. chosen aiid used throughout the work. I t was made by steeping 4j o grams of the root, sliced thin. in a liter of water for three hours at 100' C. ,1fter strain-
ing and cooling, the whites of several eggs were added aiid it was again boiled, then strained, filtered, and poured into flasks. _\ftttr thorough sterilization the infusion was ready for me. In order to get the greatest possible uiiiforniity it was foiuid desirable to make lip four or five liters at a time. In this iiiediuni tlie spores of the forins selected geriiiiiiated in from 3 to 8 hours and with great iiniforniity. grew rapidl!., and fruited normall!. (except L?oti:i,tisj in from IS to +S lionrs at a teniperature of 2s0
c.
3. ,Ifithod of Cu?tiirc. - T h e Tan Tieglieni hanging-drop culture was fonncl to be entirely satisfactor!-. T h e cylinder part, w-hich was of glass, had an interiial dianieter of I/. j mi11 and was IO. 7 niin in heiglit. This pro\-icled an abundance of os!gen for the normal development of tlie fungi. T h e cj.linders were ceiiieiited to the slip by iiieaiis of beeswax, and the covers were sealed to the cell b>-means of a ring of \-aseliiie applied to the top of the cell b!- inverting it on a glass slip covei-ed bj. a thin layer of melted vaseline. sinal1 nick \vas made ixi this ring so that when the cover n-as applied a niiiiute opeiiing iiiiglit be left through wliicli the expanding air could pass n-heti tlie cultures were placed in the thermostat at 2%' C. - i n hour later the!. were carefull!. esaiiiiiied aiid such as liad not alreacl!. beccmie hermeticall!. sealed were now macle so bj. tapping n-ith a pencil o\.er the tin?- opening referred to above. This precautioii pre\'eiits niuch trouble aiid loss when culturing n-it11 volatile si111stan ces. For conveii i en ce in h and 1i n g and e xani i ni ng 11iitle r the microscope two cells \\-ere placed on each slip. T h e cells \vert: permaiiently labeled by guninling lettered and nuiiiberecl labels to the ends of the slips. -1sa precaution against accident..; duplicate ciiltures were ne\-er placed on tlie saiiie slip. T h e various dilutions in beet infusion of tlie coiiipoiiiid to be tested were macle up in a dozen or more bottles of about 30 cc capacity. Each bottle was pro\-ided with a giass rod, clran-ii to a blunt point, by iiieaiis of which the culture drop was trail+ ferred to a cover. FOUY o r f ; w t J r y b s c f t h e .sniir~ so?iifioiz $rr( their $ln~-ed1'11 thc 6oftnm (?-the cell. T h e spores of the fuiigii.~ (;
to be tested were traiisferred froiii a pnre culture to the hangitigdrop by means of a sterile platinum needle, the utmost care being takeii to &*cz,eizf the ndhmerlzcr (f i/zr S ~ / ~ O J * C S2;1~ 0uuche.r in iiiakiiig the inoculation. T h e co\.er hearing the culture was then inverted oii the cell atid getitlj. pressed until completely closed except for the niiiiiite opeiiiiig already fully described. l17hen a set of cultures was coiiiplete all were placed in a tlieriiiostat which was kept at a coiistaiit teiiiperature of 28" C. 4. Core ~fCi.ll.7, C o z ~ r sPljkties, . etc. --After the completion of a series of cnltures all bottles, rods, etc. were thoroughly Ivaslied and placed in running water for several hours, then dried niid placed in a dr!- oven at 160' C. IYheii the cultures in the cells matured, the covers were renioved arid the cells were thorong1il~-n-aslied iiiider tlie mater tap, wiped, air-dried, and finall!placed in a drj. ol-eii at froni I I O ~ - - I Z O ~C for an lionr. T h i s gave thorongh sterilization, and at the same time drove off the last trace of an!- \.ohtile substance which iiiiglit have escaped the washing. T h e covers were first boiled in strong KOH, then in sa-era1 clianges of water. T h i s was followed by boiling in strong H9S04 K7Cr2CI7,the!. were then thoroughly rinsed, a i d again boiled in fonr Zlianges of clear water, rinsed iti 95 percent alcohol, wiped, and sterilized at 1603 C. T h e pipettes were cleaiietl bj- forcing water through tlieiii for an hour by attaching to a water tap. T h e y were then sterilized in a steam sterilizer. Ai1 occasioiial culture was found to be coiitaiiiiiiated by bacteria, cine no doubt to dnst particles bearing spores coining in contact nitli the culture in the making. Snch contamination, lion-e\-ei-,bj. bacteria or fniigi anioiiiited to less than oiie percent of tlie cultures made u p after completion of preliiiiiiiar?. ex peri iiie 11tat i on. 5. 1 ~ J ! W J * P W S S Z ~inJ Ythe ~ SGel?.- I t has long been k i i o ~ t ~ to ph!.sical chemists that ever>-liquid has a certain ~ Y Z ~ O Y ~ ~ ~ .m J - P . Previous experimenters. testing the toxic properties of aqueons solutions of various substances b>.means of the hangingdrop cultnre, have, strange to say, 01-erlooked this very important matter. This fundamental error led to much difficulty in tiianipulation and caused exceedingly erratic results. The diffi-
C S -
culties of iiiakiiig liangiiig-drop cultures coiitaiiiing, sa!., T O perceiit alcohol or potassiuiii hydroside when there is a drop of ;t'(zfcrin tlie bottom of tlie cell, \vi11 be ver!. e\.ideiit to the reader. T h e \.erJ. eiratic character of the results to lie espectetl is \\,ell shon-11 h!. a test of spores of -l~~r/~~).~p~/,~l~//i in solutions of mercuric I n a solution of 11 819200 HgCl2, tliree cultures grew and one failed : of eight cultures in i i 409600, five xren- and three failed : of ten culiures in i l Z O $ ~ O O , foiir p e n and six failed : of four cultures in I I 10240~)~ onegrew :ind tliree failed. T h i s irreg-nlarit>-of germination was doubtless diie to two factors. 1-iz. \-ariatiom in tlie size of the liaiiging-drops and \.ariatioiis in the quantity of n-ater belon. TYe1-e it possible to li:t\.e all liangitig-drops of ~ , . i - u ( . t [ i the l same size, ?lid esactl?. the same citiaiitit!. of water iii the bottom of the cells we should get i///$)riuitJi iii results. Such uiiiforiiiity. liowe\.er? nould lie iiseless. perhaps n-orse tliaii ii.seless in that it \voidd teiid to pre\wit attention being called to the fiinclaiiiental error of the iiiethod, \-iz. the me of solutions of varying i-apor pressiires i i i the saiiic cell. -1stricti?. non-\.olatile or less poisonous substance ~ v o u l c l ~ iiecessaril!-, slion- sonien-liat less variation tlian tliat sliowii 1 1 ~ mercuric chloride quoted above. The less difference there i.s lietn-eeii tlie \--ipcir-pressure of the iiiistiire in tlie lianging-droI) and that of the water belon, the slower will the error be in niaiiifesting it.?elf bJ- its effect on germinations. Of tlie \.oiatilc properties of HgCl? w e shall lia\.e more to sa!. later. In looking for a iiiethod that n-ould meet ever!. requirenieiit of tlie case, iiiaii~-preliiniiiar!. tests were macle ivitli all five fori115 of iiiolds used atid with difierent deleterious agents. Tv-o siicli tests xvith potassium cJ-aiiideare here presented in detail. It i i of interest to note that while KC?; is iiot in itself a \.olatile conipoiiiicl, in aqueous solutioii more or less h!.drolysis take.% place, resultiiig in the formatioil of a corresponding aiiioiint of the deadlj- H C S , ' which is quite volatile. Hence aqiieous soliitioiis of K C S are iii their behavior quite t!-pical of solution.< of \.ohtile coinpounds.
' F. I,. Stevens. not. Gaz. 26, 37; ( I S # ) . _. - shields. Zeit. pliys. Chenn. 12, 16; ( rS9; I .
.>
2 68
.z;
..t
e .-
'L
X
M
c; 0
r)
N
R
0
L
z
Elf,ctr ( i ( l l f i r Dis.i ocin fioii n i r d Toxic EJcct
269
~ . ~ ~ l n i i n t n i ; i ~ - C o l u i iIi gives ii the culture label b!- means of which the ciiltures \\-ere identified ; coliimn 2 the concentrations of E;C S in the hanging-drops of tlie Txrioiis ciiltiires. Column 3 gi\.es the solution - if an!. -used in the bottom of the cell. 1)rJ- implies that no water or other solution \\.as placed in the bottom of the cell. 17iitler the head ‘(germination” tlie percentage of spores germinated is given for three ohseri-ations, a t I 2 , 24, and 36 hours, respecti\-el!.. Ynder the licad developiiieiit ’* the length of tlie germ tithes of spores showing an a\.erage development is gi\.en i n niict.omilliiiieters. These data not oiil!. show that ciil tures 1iaT.ing n u f r r iu tlie bottom of tlie cells are unreliable aiid vary according to the nuioiiitt of water present, h i t that drj- ” cells are equall!- 1111satisfactor!-. Especial attention is called to the record of the ciiltiires labeled “-1IO”, Here Ive haye a solution of K C S nliich is but one-fourth the strength of that in -19 ”, the foriner, hon.e\-er, hac1 several drops of t1;e I I I 2 8 KCS solution placed in the bottom of the cell, tlie latter had water oiil!.. Tlie striking difference in the result needs no coriiiiient. Tlie results \Tit11 PciIici//i// and Ju other forill,\ abiiiidantl!- confirnied the test, aiicl lead to the conclusion that athciz i i z f c , r - is rtscd nf thc hoftoiii q/ ce/ls coii fniiiiug h n ~ i ~ < ~ - i ~ until tlie fuiig-us Iind iiiatiiretl or t h e spores iii tlie ciiltiires \vhich lint1 failed to gymiiiate, \vere transferred to piire heet infusion to test their \.italit!.. T h e perceiitag-e of spores periiiiiiatetl iii the \-arioiis ciiltitres \vas iiotetl on t \ v o occa.sioiis. first \\-lien from 20 to ;o perceiit of tliose in the checks hat1 Xcriiiiiiated :uid agaiii a f e n Iiours later. -4siinilai- iiietliod \\-as atlopte(1 to indicate tlie earl!- iii!.celial del-tlopiiicnt. - I t ,soiiie time after pmniiiatioii tlie per111 tulles slio\\-iiip mi :i\.erape der-elopiiieiit \!-ere iiieasui-et1 ant1 iiotecl : this \vas rcIienterl a fen- Iiours later, and soiiietiiiies a tliiid iiieasureiiieiit \\-a>inatle. T h e first appearaiice o f conidia \\ also iioted, 1)iit :is t l i i , ~\.er!- freqiieiitl!. occurred at iiiglit ( 11 -eel1 1 5 and 2 7 lio~i~ after s iiioculatioii ) t h e poilit \vas iiot so \vel1 noted. 111 tlie rnrlier ~ o r k the , ciiltiires i n \vhich thr. s p o r e s j r c ' i ~ t ~ io i,ijlii/o/( \yere opened at 7 2 lioiirs' aiid a num1~e-r-of the spores \\-eretraiisferretl li!. iiieaiis of a sterile platiiiuiii needle to a liaiig-iiip drop of p i r e beet iiifiision i i i a cleaii cell, in order to test \vlietlier tile!. n-err killetl bj. the agent or \\-lietliei- their periiiiti:itioii \vaq tilerel!. iiiliilii ted. If oiie pel-ceiit or iiiore siii-\.i\.ei-l tlieJ- \\-ere cla d as ~ ' / / h i h c ' t ( dif; nolie at all, or less t h a n
272
J,F. ClUi,A
oiie per cent siirvived, they were classedas ki/lcd. In most of the work, however, tlie transfers were made at 4s hours, it being found that all spores that could germinate did so in less than 36 hours, aiid usually iiiucli earlier. T h e cultures which gerininated in the presence of the cleleterious agent were likewise diI-ided into two classes : (I) those whicli, altliougli they ma>-ha\-e been retarded or stiniulated in mycelial de\~elopmeiitby tlie agents, finallj. matured a fair crop of coiiidia in about tlie normal time ; (\z) those which, although the?. germinated aiid continued to grow, presented a markedly irregular or retarded mycelial developiiieiit, aiid generally failed to fruit. Retn-een these two :lasses came - as might be supp x e d - a niunber of cultnres which it was \-ery difficiilt to classifj.. In soiiie cases there \~-ouldbe an apparentlj- noriiial iii!-celial del-elopmeiit but almost total suppression of fructification. In other cases an irregular, ineager, and even yeast-like iii!.celiuiii would cause surprise b>-fiiially developing a iiumber of apparently normal fruits. In general, a fungus was said to b e ' * injured" b y that coiiceiitratioii of tlie deleterious agent nliicli prevented its classification in class I as above. In discriminating between class 3 (inhibited spores) aiid class 4 (killed spores) attentioti is again called to the iinportance of a\-oidiiig bnrzchcs of spores in iiiakiiig the iiiocnlation. In many solutions such bunches - doubtless containing air - float on the surface of tlie drop and fail to recei1.e the full influence of the ageiit. TT'here spores from such a culture are transferred to pure beet infusion to test their \,italitj- the bunch may he brokeii u p in the process and the spores geririiiiate readily while all others are dead. X u c h can be dolie by care in inoculating, but at best it is a serions soiirce of error, a i d it has been on this accoiltit and on account of the impossibilitj- of transferring the spores ivithout taking with them sinal1 qiiantities of the agent that tlie i~dzihz'tiir~q pnz'iii rather than tlie death-point has been adopted as the ~ ~ h i ~ ~ ' c ; ~ z ' i z ' c n /in p ndiscussing iiii the experimental data. T o illustrate what has been said regarding tlie taking of notes on cultures, a typical left-hand page of tlie culture note-book [duplicate cultures omitted) is here presented. T h i s may also
U 13
help the reader to uiiderstaiid the classificatioii of the esperinieiital data presented later. T h e opposite page was aln-aj.s reserved for more esteiided notes 011 points obserL.ec1 froin tiiiie to time in the progress of the esperiiiieiit. 9. .SOIII~CC.S (If Error. - Hefore proceeding to a discussion of tlie esperiiiieiital data it iiiiglit be ne11 to ineiitioii brieflj- the soiirces of error o b s e r \ d during the preliiiiiiiar~.stud!. aiid guarded against in the progress of tlie n-ork. I . Xj-lonite cells were foiuid to ha\-e ail iiijiirioiis iiifliieiice oii some fiiiigi n-hen used for Iiaiigiiig-drop cnltures a t 2 8 ' C. 2. Bacterial contaiiiiiiatioii. 3. Lack of equilibriuiii in vapor pressures in cells. 3. Deterioration of stock solutions. ( S e e details of esperiiiient n-ith K C S . ) j. The use of impure vaseline in sealing cells. 6. r s e of spores of iiiicertaiii ag-e and qualit!-. 7 . 1-se of a culture iiiediiivi unsuited for the iioriiial developiiieiit of the fungi tested. 8. Imperfect sealing of cells. dne to the raising of the co\-er by expansion of coiitainecl air when cultures were placed in tlie th eriiios tat. 9. Transference of bunches of spores in iiioculatiiig cultures. 111. EXPERIMENTAL DATA AND DISCUSSION
Details regarding the critical poitits ( i . e. coiiceiitratioiis causing iiijiirj-s inhibition, a i d death of the various fungi i i i the more important of the tliirtj--seven compounds tested inaj- be found in the diagrams, Figs. 1-4. These give in condensed form the various specific resistances of the different molds and it is hoped that the?. will also be useful in conve).iiig to tlie reader I]!. iiieaiis of the eye a general impression as to the relative tosic powers of the 1-arious agents. I t should be distinctlj- borne in iiiiiid in consulting the diagraiiis that each 1-ertical line represents a n'nuh(l'rg of the iiuiiiber of inolecnles preseiit iii tlie solutions as you pass towards tlie right, the whole space between two
iwtical liiies representing one concentration. I11 tlie diagi-aiii.~ Iiorizontal daslies denote that the iiiold \vas killed those colicentrations : a coiitiiiucous horizontal line denote? inliibitioii ( i f tlie spores : a clotted liiie c1eiiote.q iii,jiir!., nhile altei-iiatiii,y dot, ;tiid daslies iiidicate that tlie solutions permitted practicall!. iioriiinl cle\-elopiiieiit. ;Is has previousl!. heeii stated, the clii.icliiig liiit. hetn.eeii the.se last t\vo classes is not sharp and, in some c:ist..\ tlierefore, t n o sets of liiies are used for a gil-eii conceiitratioii. T(i make the diagraiiis a little clenrer tlie most dilute solutioii.~ that >.et kill are connected 1 ) ~ .a liiie ; the saiiie is dolie n-itli tlic iiiost diliite solutions, that !.et iiiliihit and n-itli the iiiost ciiliitc. solutions that !-et cause iiijur>.. T h e letters in :I xwtical l i i i t a t the left stand for tlie different fiiiig-i, -I3 S,(3. I:, and I' r v p reseli ti iig - l.ykr- non-toxic having like C1 ‘
Iiahlenherg and True, 1896, loc. cit. 1.’
92.
J. E.: Clff7-k
284 -
and SO3less than I 32 the toxic value of ionic H. T h e dissociation of H2S04at the inhibiting point is about 62 percent only.' Each IOO molecules, then, at this concentration, break up into
-
-
-
approximatelj- 124 H, 76 HS04,and 23 SO4 ionr. T h i s solution having a greater toxic value than a similar concentration of ionic H, the excess of toxic properties must be dne to the partially dissociated group, HSO,. . ! , P referring to Table I. it will lie seen that the toxic value of this anion is approsimatelj~1.3 t
in terms of ionic H. In this as in the other mineral acids, .Sf~i-z,o.J/1rrfcic:iistzs showed the greatest resistance, / I '2 being necessary to kill. IIot/y,tis \vas the most sensitive, 71 '16H2S04lieiiig f a t d . Aei'i~-A c i d , CH~.COOH, 2j.6- 8;,314. This acid at the inhibiting point, 72,'2;, is but 2 rercent dirsociated.' T h e toxic value of tlie anion \vas foiuicl to be about I 13 H, but assosmall a portion oi tlie acid is dissociated tlie influence of the anion may lie disregarded. T h e properties of this acicl are therefore to be attribiited almost solely to the undissociated iiioleciile,
CH7.CC)OH,\vhich proves to have a toxic value of 2.8 H. I+/iir/'//ii~/u shoned a marked specific resistance to this acid, reqiiiring 11 '2 for 4s hours to kill. This obser\-ation was accepted on1~-after repeated trials. Bofi;lgtis, on the other hand, \vas particularll- susceptible, 72, 64 being fatal. T h a t acetic acid shoiild prove so inucli more toxic to fungi than the mineral acids n-as not aiiticipated.3 Heald, fonnd that it had but I S the toxic value of H 011 2 ' ~ l r and ~ I 4 on Pisum7 while Kahlenberg atid Trues fonud ahout the satlie relation with Lzijl'iizrs. Tlie great \.ariation in protoplasmic resistance to this acid is slio\vii by the folloniiig data : T h e 1-inegar eel - A'hn(idit2's Ko1ilr:inscli. IYiecl. . i n t i . 26, 196 ISSj 1. Illid. 26, 197 ( 1 8 S j I . 3Zigula. Bot. Central. 41, 20; (1S90). I:. I>.Healcl. iS96, loc. cit. p . r j o . .' Ka1ilenl)erg :uitl True. rS96. loc. cit. p. I I O .
1
nrrti- thril-es in a 71 ’2 solution, which is fatal to Prrii(.i//i///r/ spores. , J s $ c ~ ~ i / / z spores ~s are killed b!- rz/8 and those of Z ? o t ~ ; i ~ tis by 12/64 ; 1z,!203 is fatal to %cn, I / ‘1630 to Pikuru. I t is of interest to note in this connection that not onl!- are great differences to be expected bet\r-een different organisnis in their resistance to deleterious agents, hiit different individuals o f the same genus and species may have \.er!- different powers of resistance, depending Iar,oel>,no doubt on pre\,ious en\.ironineiit. Pfeffer,’ for instance, grew z4s$v~/;q-i//zf.r on a nutritive medium containing 8 percent dextrose and one percent acetic acid, and found that the fungus assimilated a far larger ainoiint of the acid than of the dextrose. I t will be noted that a / S or 0. j percent acetic acid proved fatal to the spores of ,4~ykr;yi//u.s used in tliir; stud!-, and in a 0.1j percent solution less rliaii one percent germinated. Thc C~h/or*ncctic A4(-ids. These acids are foriiied 11;. replacing one, t\vo, and three atoms of H, respectivel!., in the acetic acid radical 1)~. the elenlent C1 ; thus, H O H-(!-C-OH
I
H O
-icetic acid.
O
1
:
H--C‘-C-OH
CI-C-b-OH
I
I
c1
H
H
C1
3Ionocliloracetic a. the percentage of
I I i
Y
i
..
2 94
J. F. C/nr,k
undissociated molecules, gives in ternis of ionic H the toxic value of tlie different acid molecules. Part I1 of this table is worked out similarly and has reference to the toxicity of tlie acids toviards the iiiolds as showi by their power to hi//the spores. X11 acids tested retard germination atid early growth of the inold fungi. T h i s retarding influence was evident in greater dilution with the tnineral than with the acetic acids. Cultures not greatly retarded almost invariablj- take on new life not many hours after germination, and presentlj. 01-ertake and siirpass the checks iii n~j.celial development. This excessive development of ~ i i y e l i u i i i iisiiall!- reaches its iiiasiiiiniii in ciiltiires two or three remox-es froiii the concentration inhibiting germination, and is usually nccoiripanied by retardation of fruiting. In other words, I 4 to 1'8 the 1 e t M concentration 1ias a strong stimulating influence 011 tlie n i ~ ~ e l i ade\.eIopinent, l and teiids to snppress or at least retard fructificatioii. Siiialler quantities of acid proved on an average to be injurious to E d o r e j h n l u i i z than to the other forms, 20,/204Snormal beiiig on the a\-erage distiiictl!- detri~iiental. T h e coefficients of tlie other foriiis were as follows : Botiy~iis,23 ; Perzici//iztiiz, Z S ; ~-l.~pci~yi//its, 4 2 ; Ster,icyiiintorlisfis~64. G?docij/ln/iti/1was also tlie most easily inhibited, its relatii-e inliibiting coefficient being 7 2 , folloned b!- Botq~L'is,76 : &uic i / / i i f ~ r i ~T O O : L4.$ci;qi/li/.s, 104 ; and Sfrr-z~7iinfoc?isti~~, 200. 110t7ytis, however, was tlie most easily killed. T h e order in this respect was Bofr:i~tis,I O O : ~ d O C f ' $ / I n / i / i J 1 ,137 ; A.@cr*gi'll~ts,2 7 2 ; S t c ' i ' ~ ~ i r i r r t t i L ~ ~ ~ . s369 t i . ~ ~; , and final1j- Penii-illiii~r~, ivhose spores showed by far the greatest resistance, represented by the coefficient 498. This high resistance of I1t'iiicz/Iiiti~~ in regard to the death-point ma!- be partly due to the tendency of the spores to adhere iii bunclies in making tlie inoculation, a difficult!- never fully oi-ercome. T h e lieariiig of these results 011 tlie modern theories of the chemical and pliysiological action of substances in aqueous solution is of sufficient iiiiportance to demarid a brief treatment.
Elcl-tro(i~fi~. D i . ~ , ~ o c i ~oitd ~ t i oT(i.ri(. ~ ~ Ef(9l-t
2 9paper i n 1895. I,iiie I g-i~;esthe relati\-e cli.~sociationof iiorliial solutions of these acids (escept H C S 1, HCl being taken as a staiidarcl as it is i i i all the tehts recorded iii this table. Line 2 g i \ e the relati\-e pair ers of tlie different acids to promote the \vell-kiion-ii catalj.sis 11f aqiicous solutioiis of iiir:tli>.l acetate. Line 3 gi\.es tlittir relati\.? activity in clecoiiiposiiig calciiuii oxalate. Line 4 gii-es 0.t\\aid's determiiiatioiis of their relative affinities fur Ii?.drosidt.s. Line 5 gix-es their relative acti\.ity in iiix-ertiiig cniie sugar. The corresporideiice 1xtn.eeii the figures iii these 61-e l i i i e i.j certainly quite striking, especia11!- \\.lien it is renieiliberecl that the bolutioiis used in the tliffereiit tests n.ere not of tlie :.'1;111 ' c concentration. ;Illo\vr.iice Iiiiist also be made for the e s p - i iiieii tal difficulties iiir-ol\-ecl iii cheiiiical iiiea~ureiiients. Urider these! circunistaiices it is iiot strange that iiiaii!. cheiiiists c;~iiit' to the coiiclii.\ioii that it is thc i o i c i z c ~ dj m - (I/' ~ (z tili.soi;mcti .icf/,x f u / ~ riild l ~ ~ f~h n i c i i c ( 1 ' n.liicli is cheinicall!- a d \ - e . T o quote fro111 1\7ietIiaiii :i 11-eiimy tal;? i t , tlieii. that 0111~-that portion of ;L lied!, i.s clieinicall?- acti7;e ~~-1iicIi is electrol!~ticall~.acti1.e - t h a t ionization is iiecessar!. for clieinical activitj- jnst as it is iiece,-sary for electrol!.tic conducti\.it!.." 1T7iat applies to clieinical actix-ity iiiiist also a1)pl!. to physic,logical activit?. for in its iiltiniate aii:il>+ tlie latter is dollljtle..,, 4rliieto the foimier. Kali1enl)ei-g and ' h e + 1-ei11a1-1~~I t h:is :I]\va!.s liecii takeii as asionistic that tlie pli~.siologicalactio11 vf ,an\- sulistmice is diie to its cheiiiical cliaracter." b L
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.irrlieiiii:~, Keclierclies stir la coiitluctivit6 gal\-,iiiicjue lies Stockllolrn , ;ss; ". ' -1.1 , e v ~ - Zeit. pliya. Clierii 17, 31; (xSgLi I , .' IV. C.1). II~liethatii.Inc. cit. 11. 16j. ' I
